First non-ATP competitive glycogen synthase kinase 3 β (GSK-3β) inhibitors: Thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer's disease

Article abstract of DOI:10.1021/jm011020u

Glycogen synthase kinase 3 β (GSK-3β) has a central role in Alzheimer′s disease (AD). Selective inhibitors which avoid τ hyperphosphorylation may represent an effective therapeutical approach to the AD pharmacotherapy and other neurodegenerative disorders

Full text of DOI:10.1021/jm011020u

1292
J . Med. Chem. 2002, 45, 1292-1299
F ir st Non -ATP Com p etitive Glycogen Syn th a se Kin a se 3 â (GSK-3â) In h ibitor s:
Th ia d ia zolid in on es (TDZD) a s P oten tia l Dr u gs for th e Tr ea tm en t of Alzh eim er ’s
Disea se
Ana Martinez,*,† Mercedes Alonso,† Ana Castro,† Concepcio´n Pe´rez,† and Francisco J . Moreno‡
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, 28006 Madrid, Spain, and Centro de Biolog´ıa Molecular
“Severo Ochoa” (CSIC-UAM), Universidad Auto´noma de Madrid, 28049 Madrid, Spain
Received August 13, 2001
Glycogen synthase kinase 3 â (GSK-3â) has a central role in Alzheimer’s disease (AD). Selective
inhibitors which avoid τ hyperphosphorylation may represent an effective therapeutical
approach to the AD pharmacotherapy and other neurodegenerative disorders. Here, we describe
the synthesis, biological evaluation, and SAR of the small heterocyclic thiadiazolidinones (TDZD)
as the first non-ATP competitive inhibitor of GSK-3â. Their synthesis is based on the reactivity
of sulfenyl chlorides. In GSK-3â assays, TDZD derivatives showed IC50 values in the micromolar
range, whereas in other protein kinases assays they were devoid of any inhibitory activity.
SAR studies allowed the identification of the key structural features. Finally, a possible
enzymatic binding mode is proposed.
In tr od u ction
â-amyloid-induced neurotoxicity and presenilin-1 muta-
tion pathogenic effects.10,11 These observations suggest
a central role for GSK-3â in AD.
Neurofibrillary tangles (NFTs) are one of the char-
acteristic neuropathological lesions of Alzheimer’s dis-
ease (AD) and other neurodegenerative processes such
as frontotemporal dementia, Pick’s disease, progressive
supranuclear palsy, and corticobasal degeneration.1
They are composed of a highly phosphorylated form of
the microtubule-associated protein τ.2 τ, in the adult
human brain, is a set of six protein isoforms, whose
binding affinity to microtubules can be modulated by
phosphorylation. A total of 25 sites of aberrant phos-
phorylation has so far been identified in τ protein. Many
of these sites are serine or threonine residues that are
immediately followed in the sequence by proline resi-
dues and, hence, are candidate phosphorylation sites for
proline-directed kinases.3
Human GSK-3 is a ubiquitously expressed serine/
threonine protein kinase implicated in the regulation
of many physiological responses in mammalian cells by
phosphorylating a variety of cytoplasmic and nuclear
proteins.12 It also phosphorylates components of the
neuronal cytoskeleton including the microtubule-associ-
ated protein τ and neurofilament heavy chain. Two
nearly identical forms of GSK-3 exist: GSK-3R and
GSK-3â. Both are constitutively active in resting cells,
but their differential expression and the lack of relation
between transcription and translation in some tissues
indicate that GSK-3R and GSK-3â have different means
of regulation.13
Advances in genetics and transgenic approaches have
a continuous impact on our understanding of AD and
related disorders, especially since aspects of the histo-
pathology and neurodegeneration can be reproduced in
animal models. The recent transgenic mice overexpress-
ing GSK-3â in the brain during adulthood are a valuable
tool for the study of the pathogenesis and potential drug
therapy of AD.14
Recently, the 3D crystalline structure of GSK-3â has
been described15,16 and has now begun to illuminate a
sophisticated regulatory mechanism which is capable
of processing different signals within the same cell.17
These results may also lead to the design of function-
specific drugs that disrupt some actions of GSK-3â but
leave others intact.18
There is no doubt that potential selective inhibitors
of GK3â which avoid τ hyperphosphorylation may
represent an effective new therapeutical approach to the
treatment of AD. Up to now, few compounds are known
with this inhibitory enzymatic property. Lithium be-
haves as a specific inhibitor of the GSK-3 family of
protein kinases in vitro and in intact cells,19 and recent
studies suggested a possible role of lithium in the
There is some evidence to suggest that τ hyperphos-
phorylation is an early event in the development of
neurofibrillary pathology. Thus, the blockade of this
hyperphosphorylation step may be a prime target at
which to interrupt the pathogenic cascade.4, 5
Although many protein kinases are known to phos-
phorylate τ in vitro,6 the in vivo players contributing to
the hyperphosphorylation of τ remain elusive. Recent
studies have demonstrated that glycogen synthase
kinase 3 â (GSK-3â) and cyclin dependent kinase 5
(cdk5) most readily contribute to the ATP-induced “AD-
like” phosphorylation of τ,7 while studies on transfected
cells and on cultured neurones showed that GSK-3â
activity controls τ phosphorylation and τ functional
interaction with microtubules.8 GSK-3â binds to pres-
enilin 1 and plays a role in Wnt and insulin signaling
cascades, both of which have been proposed to be linked
to AD.9 Moreover GSK-3â activity may be altered in AD
brain, and it has been postulated that GSK-3â mediates
* Address correspondence to Dr. Ana Martinez. Tel: 91-5622900.
Fax: 91-5644853. E-mail: iqmam06@pinar2.csic.es.
† Instituto de Qu´ımica Me´dica (CSIC).
‡ Centro de Biolog´ıa Molecular “Severo Ochoa” (CSIC-UAM).
10.1021/jm011020u CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/13/2002

First Non-ATP Competitive GSK-3â Inhibitors
Ch a r t 1. Inhibitors of GSK-3â
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1293
Sch em e 1a
a
Reagents: (i) Cl2/hexane/N2/-15 °C; (ii) R′-NdCdO/hexane/
N2/rt; (iii) air/rt.
Sch em e 2a
inhibition of GSK-3-induced τ phosphorylation.20 How-
ever, its high milimolar inhibitory concentration pre-
vents lithium from its potential therapeutical use.
Some compounds as bisindole21 or anilino22 maleim-
ides, kenpaullone,23 indirubin,24 or the marine natural
product hymenialdisine25 (Chart 1) have recently been
reported as GSK-3â inhibitors. All of them compete
reversibly with ATP in their GSK-3â inhibition, showing
a wide protein kinase inhibition profile. Here, we
describe the small heterocyclic thiadiazolidinones (TDZD)
as the first non-ATP competitive GSK-3â inhibitors as
promising candidates for further development of AD
pharmacotherapy.26 Their synthesis, structure-activity
relationships, and a hypothetical binding mode will be
described.
a
Reagents: (i) Cl2/hexane/N2/-15 °C; (ii) Et-NdCdS/hexane/
N2/rt; (iii) air/rt.
cyanate to the corresponding iminosulfenyl chloride. To
check this hypothesis, chlorination of benzylisothiocy-
anate and subsequent reactions with different alkyl-
isothiocyanates were assayed. In all cases, a complex
reaction mixture was obtained, and only the thione
benzylated derivative 23 was isolated after several
chromatographic purifications. Only when ethylisothio-
cyanate was used as the second reagent was the thione
derivative 24 obtained as the minoritary product in low
yield (Scheme 2). These results led us to discard the first
proposed reaction mechanism. The reactivity of imino-
sulfenyl chlorides with isothiocyanates is lower than
with isocyanates, and probably the thione derivatives
21-23 result from autocondensation of the correspond-
ing iminosulfenyl chlorides.
Ch em istr y
Thiadiazolidinones 1-20 were synthesized following
a pathway which is based on the reactivity of N-alkyl-
S-[N′-(chlorocarbonyl)amino]isothiocarbamoyl chlorides
with isocyanates.27 This synthesis was previously used
in our laboratory for the efficient preparation of potas-
sium channel openers28 and cholinergic drugs.29,30 Thus,
chlorination of N-alkyl or N-arylisothiocyanates in an
inert atmosphere and subsequent reaction with N-alkyl
or N-arylisocyanates produces sparingly soluble 3-oxo-
thiadiazolium salts via the intermediate iminochloro-
methylsulfenyl chloride. These heterocyclic salts are
exceptionally reactive, and in the presence of moist air,
it was possible to obtain through hydrolysis the 1,2,4-
thiadiazolidine-3,5-diones 1-20 as white crystalline
solids after evolution of hydrogen chloride (Scheme 1).
In all the reactions assayed, a minoritary product could
be detected by TLC which could be isolated by silica gel
column chromatography only in some cases. The struc-
tural elucidation by analytical and spectroscopic (1H and
13C NMR) data revealed a 5-oxo-1,2,4-thiadiazolidin-3-
thione chemical structure for the minoritary derivatives
21-23.
Introduction of an imino moiety at the 5-position of
the thiadiazolidine framework was achieved by the basic
hydrolysis of the urea derivative 26 (Scheme 3). This
last compound was easily obtained following a described
procedure of 5-aminophenyl-1,2,3,4-thiatriazole rear-
rangement with isocyanates and isothiocyanates31
(Scheme 3).
The structure of all new compounds was elucidated
from their analytical and spectroscopic data (1H and 13C
NMR) which are collected in the Experimental Section.
Unequivocal assignment of all chemical shifts (1H and
13C NMR) was done using bidimensional experiments
such as HMQC for one bond correlation and HMBC for
long distance proton/carbon correlation. Thus, N2-CHx
correlated exclusively with the quaternary carbon C-3,
while N4-CHx correlated with C-3 and C-5 heterocyclic
carbons, providing an unequivocal structure assigna-
tion.
The formation of these thione compounds could
initially be explained by addition of the starting isothio-

1294 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Martinez et al.
Ta ble 2. IC50 Values for Kinase Inhibition of TDZD 8
Sch em e 3a
protein kinases
IC50 (µM)
specific activities
Cdk-1/cyclin B
CK-II
PKA
>100
>100
>100
>100
0.4 nmol Pi/min/mg protein
0.4 µmol Pi/min/mg protein
4 nmol Pi/min/mg protein
0.5 nmol Pi/min/mg protein
PKC
a
Reagents: (i) Et-NdCdO/Et3N/dry THF/70 h/rt; (ii) EtCO2-
N-CdS//Et3N/dry THF/70 h/rt; (iii) NaOH/MeOH/10 h.
Ta ble 1. Glycogen Synthase Kinase-3â Inhibition of
Thiadiazolidinones 1-28
F igu r e 1. Double-reciprocal plot of kinetic data from assays
of GSK-3â protein kinase activity at different concentrations
of TDZD 8. GS-1 concentrations in the reaction mixture varied
from 6.5 to 100 µM, TDZD 8 concentrations are 3.3 and 10
µM, and the concentration of ATP was kept constant at 15
µM. V is picomoles of phosphate/20 min.
compd
R
R′
X
Y
IC50 (µM)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25b
26c
27
28c
Et
Et
Et
Et
Me
Et
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
5
25
10
35
10
70
>100
2
Pr
iPr
Et
cyclohex
Et
nBu
cyclohex
Bn
Me
Me
Et
enzyme activity. As it is shown, a wide range of TDZD
inhibits GSK-3â with IC50 values in the micromolar
range.
Bn
Bn
Ph
7
10
2
3
4
6
4
4
6
5
2
3
20
8
10
6
10
75
65
100
Bn
Me
Me
Me
Me
Me
Me
Me
Me
Me
Selectivity of protein kinase inhibition33 is critical for
pathway analysis in cellular systems and to avoid
widespread effects in a potentially therapeutical treat-
ment. For these reasons, TDZD 8 was assayed against
other kinases as protein kinases A and C (PKA and
PKC), casein kinase II (CK-II), and cyclin dependent
kinase 1 (Cdk-1/cyclin B). Although a number of recent
articles23-25 described potent inhibitors of Cdk-2 which
also inhibit GSK-3â, all of the TDZD derivatives here
evaluated showed IC50 values of >100 µM (see Table
2). These results point to a specific GSK-3â inhibition
probably out of the ATP binding region of the enzyme.
4-BrPh
3-BrPh
2-BrPh
4-ClPh
4-FPh
4-CF3Ph
4-MePh
4-OMePh
1-naphthyl Me
Et
Et
Ph
Bn
Et
Me
Et
Ph
Bn
Bn
Et
Ph
Ph
Ph
S
S
S
S
O
O
S
N-3Pyr
NCONHEt
NH
To investigate the mechanism of TDZD’s action on
GSK-3â, several kinetic experiments were performed.
First, we varied both GS-1 (6.5, 10, 15, 25, 50, and 100
µM) and TDZD 8 concentrations (3.3 and 10 µM) with
a constant concentration of ATP (15 µM). Double-
reciprocal plotting of the data (Figure 1), in which each
point is mean of two different experiments, suggests
that TDZD 8 acts as a noncompetitive inhibitor of GS-1
binding.
Et
CO2Et
NCO2Et
a
IC50
: 50% inhibitory concentration of glycogen synthase
kinase-3â activity (µM). Assays were performed in triplicate.
b
Reference 28. c Reference 31.
Biologica l Resu lts a n d SAR
The GSK-3â activity of the new TDZD derivatives
here synthesized (1-27) was determined following a
method described in the experimental part. Briefly,
GSK-3â enzyme (Sigma) was incubated with ATP and
GS-1 as substrate32 in the presence and in the absence
of the corresponding test compound. Finally, the GSK-
3â activity was expressed in picomoles of phosphate
incorporated per 20 min of incubation or in the percent-
age of maximal activity.
Moreover, kinetic experiments varying both ATP
levels (6.5, 10, 15, 25, 50, and 100 µM) and inhibitor
concentrations were performed. In these cases, two
different GSK-3â inhibitors were used: our TDZD 8 and
the known ATP competitive inhibitor Ro 31-8220.21 We
have measured the kinase activity increasing ATP up
to 100 µM where enzyme saturation could be ensured.
Double-reciprocal plotting of the data (Figure 2), in
which each point is mean of two different experiments,
suggests that TDZD 8 act as a noncompetitive inhibitor
Results are collected in Table 1 where IC50 represents
the compound concentration that inhibits the 50% of the

First Non-ATP Competitive GSK-3â Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1295
F igu r e 3. Proposed binding mode of TDZD to GSK-3â.
On the other hand, the nature of the substituent of
N4 is also important for inhibition. When comparisons
allow, potency is always enhanced with the introduction
of an aromatic moiety, such as phenyl, methylphenyl,
or naphthyl groups (compounds 8 and 11-20), suggest-
ing favorable hydrophobic interactions with the protein.
Regarding the alkyl substituents in this N4 position,
only the smallest ones, such as ethyl moiety, result in
a GSK-3â inhibitor (compounds 1 versus 6 or 7).
Substitution of the carbonyl group attached to the C3
position of the heterocycle by a thiocarbonyl fragment
led to derivatives that mainly retain the inhibitory
potency (compounds 21, 23, and 24 versus 2, 10, and 9,
respectively) but decreased compound solubility and
stability. However, when the C5 carbonyl group is
replaced by the bioisosteric imino moiety, a dramatic
decrease in the inhibition is observed (compounds 25-
28). This fact might suggest that the C5-carbonyl group
is critical for binding.
During the preparation of this manuscript, the 3D
crystalline structure of GSK-3â has been reported by
two different groups.15,16 In both cases, an oxyanion
binding site was determined, and a small molecule
(HEPES or phosphate) is necessary for the formation
of crystals and the stabilization of the active conforma-
tion. According to these authors, the convergence of
three basic residues (Arg 96, Arg 180, and Lys 205) is
required for the active conformation, and it can only be
achieved by counterbalancing negative charge provided
by the oxyanion. Regarding the chemical structure and
the SAR observed in the TDZD-specific GSK-3â inhibi-
tors here described, we propose a hypothetical binding
mode that must be further theoretically and experimen-
tally confirmed. In this model, the negative charge on
the TDZD heterocycle may be the driving force to
recognize the oxyanion binding site of the enzyme.
Moreover, interactions between the side chain of resi-
dues Arg 96 and Lys 205 and the crucial carbonyl groups
of TDZD might be possible (Figure 3). An additional
disposition of the inhibitor with the N4 pointing out to
the inside of the activation loop toward Tyr 216 might
be explain the potency of the N4-aryl TDZD by potential
π-π nonbonded interactions.
F igu r e 2. Double-reciprocal plot of kinetic data from assays
of GSK-3â protein kinase activity at different concentrations
of inhibitors. ATP concentrations in the reaction mixture
varied from 6.5 to 100 µM, and inhibitor concentrations are
3.3 and 10 µM for TDZD 8 and 2 and 5 µM for Ro 31-8220.
The concentration of GS-1 was kept constant in both experi-
ments at 15 µM. V is picomoles of phosphate/20 min.
of ATP binding while the ATP competitive binding mode
of Ro 31-8220 is shown.
These results provide the first non-ATP competitive
inhibitors of GSK-3â. To date, all the GSK-3â inhibitors
described compete with the ATP in their binding mode
to the enzyme. The property here described for TDZD
inhibitors could represent an important hit in their
potential use in pharmacotherapy.
Taking into account the interesting enzymatic profile
found in these TDZD compounds, preliminary structure-
activity relationships can be done.
Con clu sion s
The size of the substituent attached to the N2 of the
thiadiazolidinone ring seems to be crucial for GSK-3â
inhibition, the methyl moiety being the best substitution
obtained in this test set (see compound 1 versus 2-5,
or TDZD 8 versus 9 and 10). This fact could point to a
steric hindrance in the enzyme.
An efficient synthetic approach based on the reactivity
of sulfenyl chlorides to derivatives of a novel kinase
inhibitor template, the thiadiazolidin-3,5-dione (TDZD)
derivatives, is described. These compounds represent
the first non-ATP competitive inhibitors of GSK-3â
reported to date. Preliminary structure-activity rela-

1296 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Martinez et al.
7.3 Hz); 1.24 (t, 3H, CH2CH3, J ) 6.9 Hz); 1.61 (m, 2H,
CH2′CH2CH3); 3.71 (c, 2H, CH2CH3, J ) 6.9 Hz); 3.9 (t, 2H,
′CH2CH2CH3, J ) 7.3 Hz). 13C NMR (CDCl3): 2.2 (CH2-
CH2′CH3); 13.1 (CH2CH3); 22.1 (CH2′CH2CH3); 39.2 (′CH2CH2-
CH3); 46.5 (CH2CH3); 152.2 (3-CdO); 166.1 (5-CdO). Anal.
(C7H12N2SO2) C, H, N, S.
4-Eth yl-2-isop r op yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (4).
Reagents: Ethyl isothiocyanate (0.56 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), isopropyl isocyanate (0.63 mL, 6.5
mmol). Conditions: rt, 9 h. Purification: silica gel column
chromatography using AcOEt/hexane (1:3). Yield: 0.37 g (30%)
as yellow oil. 1H NMR (CDCl3): 1.20 (d, 6H, CH(CH3)2, J )
6.6 Hz); 1.31 (t, 3H, CH2CH3, J ) 6.2 Hz); 3.73 (c, 2H, CH2-
CH3, J ) 6.2 Hz); 4.62 (m, CH(CH3)2). 13C NMR (CDCl3): 13.1
(CH2CH3); 21.2 (CH(CH3)2); 37.4 (CH(CH3)2); 46.9 (CH2CH3);
152.2 (3-CdO); 166.1 (5-CdO). Anal. (C7H12N2SO2) C, H,
N, S.
2-Cycloh exyl-4-eth yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (5)
a n d 2,4-Dieth yl-5-oxo-1,2,4-th ia d ia zolidin e-3,5-d ion e (21).
Reagents: Ethyl isothiocyanate (0.56 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), cyclohexyl isocyanate (0.825 mL, 6.5
mmol). Conditions: rt, 10 h. Purification: silica gel column
chromatography using AcOEt/hexane (1:10). Yield: The first
fraction yielded 0.12 g of 5 (2%) as yellow oil. 1H NMR
(CDCl3): 1.20 (t, 3H, CH2CH3, J ) 7.1 Hz); 1.31 (t, 3H,
CH2′CH3, J ) 7.2 Hz); 3.33 (c, 2H, ′CH2CH3, J ) 7.2 Hz); 3.89
(c, 2H, CH2CH3, J ) 7.1 Hz). 13C NMR (CDCl3): 12.2 (CH2CH3);
15.7 (CH2′CH3); 42.4 (CH2CH3); 45.8 (′CH2CH3); 146.3 (3-CdS);
168.2 (5-CdO). Anal. (C6H10N2OS2) C, H, N, S.
The second fraction yielded 0.73 mg of 21 as white solid
(49%); mp ) 45-48 °C. 1H NMR (CDCl3): 1.20 (t, 3H, CH2CH3,
J ) 7.1 Hz); 1.31-1.92 (m, 5H, chex); 3.72 (c, 2H, CH2CH3,
J ) 7.1 Hz). 13C NMR (CDCl3): 13.0 (CH2CH3); 39.8 (CH2CH3);
24.7; 25.1; 31.73; 53.71 (C chex); 152.2 (3-CdO); 166.2
(5-CdO). Anal. (C10H16N2O2S) C, H, N, S.
tionships show the key features for inhibition and have
been used to propose a hypothetically enzymatic binding
mode. The potency and the selectivity of TDZD should
allow their use as tool compounds to aid resolution of
the complex signaling pathway where GSK-3â is impli-
cated and as new drugs for the effective treatment of
Alzheimer’s disease and other neurodegenerative pro-
cess in which the τ protein is involved.
Exp er im en ta l Section
Ch em ica l P r oced u r es. Melting points were determined
with a Reichert-J ung Thermovar apparatus and are uncor-
rected. Flash column chromatography was carried out at
medium pressure using silica gel (E. Merck, Grade 60, particle
size 0.040-0.063 mm, 230-240 mesh ASTM) and preparative
centrifugal circular thin-layer chromatography (CCTLC) on a
circular plate coated with a 1 mm layer of Kieselgel 60 PF254,
Merk, by using a Chromatotron with the indicated solvent as
eluent. Compounds were detected with UV light (254 nm). 1H
NMR spectra were obtained on Varian XL-300 and Gemini-
200 spectrometers working at 300 and 200 MHz, respectively.
Typical spectral parameters were as follows: spectral width
10 ppm, pulse width 9 µs (57°), data size 32 K. 13C NMR
experiments were carried out on the Varian Gemini-200
spectrometer operating at 50 MHz. The acquisition parameters
were as follows: spectral width 16 kHz, acquisition time 0.99
s, pulse width 9 µs (57°), data size 32 K. Chemical shifts are
reported in δ values (ppm) relative to internal Me4Si, and J
values are reported in hertz. Elemental analyses were per-
formed by the analytical department at C.N.Q.O. (CSIC), and
the results obtained were within (0.4% of the theoretical
values.
Gen er a l P r oced u r e for th e Syn th esis of 1,2,4-Th ia d i-
a zolid in e-3,5-d ion e. Chlorine was bubbled slowly through a
solution of aryl or alkyl isothiocyanate in dry hexane (25 mL),
under nitrogen atmosphere, at -15 °C to -10 °C. Chlorine
was generated by the addition of 35% HCl to KMnO4. The
temperature of the reaction mixture was carefully controlled
during the addition step. At this point, the N-aryl or N-alkyl-
S-chloroisothiocarbamoyl chloride was formed. Afterward,
alkyl or aryl isocyanate was added. The mixture was stirred
at room temperature between 8 and 10 h. After this time, the
resulting product was purified by suction filtration and re-
crystallization or silica gel column chromatography using the
appropriate eluent. In some cases, it was possible to isolate
5-oxo-1,2,4-thiadiazolidine-3-thione as secondary product as
is indicated below.
4-n -Bu tyl-2-eth yl-1,2,4-th iadiazolidin e-3,5-dion e (6). Re-
agents: n-Butyl isothiocyanate (0.78 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), ethyl isocyanate (0.51 mL, 6.5 mmol).
Conditions: rt, 8 h. Purification: silica gel column chroma-
tography using CH2Cl2/hexane (1:1). Yield: 0.51 g (44%) as
yellow oil. 1H NMR (CDCl3): 0.92 (t, 3H, CH2CH2CH2CH3,
J ) 7.14 Hz); 1.25 (t, 3H, CH2CH3, J ) 7.14 Hz); 1.32 (m, 2H,
CH2CH2CH2CH3); 1.55-1.72 (m, 2H, CH2CH2CH2CH3); 3.65
(t, 2H, CH2CH2CH2CH3, J ) 7.14 Hz); 3.66 (c, 2H, CH2CH3,
J ) 7.14 Hz). 13C NMR (CDCl3): 13.3 (CH2CH2CH2CH3); 13.5
(CH2CH3); 19.5 (CH2CH2CH2CH3); 29.5 (CH2CH2CH2CH3); 39.7
(CH2CH3); 42.1 (CH2CH2CH2CH3); 152.7 (3-CdO); 165.7 (5-
CdO). Anal. (C8H14N2SO2) C, H, N, S.
4-Cycloh e xyl-2-m e t h yl-1,2,4-t h ia d ia zolid in e -3,5-d i-
on e (7). Reagents: Cyclohexyl isothiocyanate (0.78 mL, 6.5
mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl isocyanate
(0.38 mL, 6.5 mmol). Conditions: rt, 10 h. Purification: silica
gel column chromatography using AcOEt/hexane (1:10).
Yield: 0.045 g (3%) as yellow oil. 1H NMR (CDCl3): 1.25-
2.20 (m, 5H, chex); 3.1 (s, 3H, CH3).13C NMR (CDCl3): 24.9;
25.8; 28.8; 55.0 (C chex); 31.3 (CH3); 153.4 (3-CdO); 165.7 (5-
CdO). Anal. (C9H14N2SO2) C, H, N, S.
4-Eth yl-2-m eth yl-1,2,4-th iadiazolidin e-3,5-dion e (1). Re-
agents: Ethyl isothiocyanate (0.56 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), methyl isocyanate (0.38 mL, 6.5
mmol). Conditions: rt, 8 h. Purification: CCTLC using AcOEt/
hexane (1:1). Yield: 0.30 g (29%) as orange oil. 1H NMR
(CDCl3): 1.22 (t, 3H, CH2CH3, J ) 6.9 Hz); 3.12 (s, 3H, CH3);
3.71 (c, 2H, CH2CH3, J ) 6.9 Hz). 13C NMR (CDCl3): 13.1
(CH2CH3); 31.3 (CH3); 46.5 (CH2CH3); 153.2 (3-CdO); 165.5
(5-CdO). Anal. (C5H8N2SO2) C, H, N, S.
4-Ben zyl-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (8)
a n d 2,4-Diben zyl-5-oxoth ia d ia zolid in e-3-th ion e (23). Re-
agents: Benzyl isothiocyanate (0.86 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), methyl isocyanate (0.38 mL, 6.5
mmol). Conditions: rt, 8 h. Isolation (8): filtration of reaction
mixture. Purification: recrystallization from hexane. Yield:
2,4-Dieth yl-1,2,4-th iadiazolidin e-3,5-dion e (2). Reagents:
Ethyl isothiocyanate (0.56 mL, 6.5 mmol), 35% HCl (3.1 mL),
KMnO4 (0.5 g), ethyl isocyanate (0.51 mL, 6.5 mmol). Condi-
tions: rt, 8 h. Purification: silica gel column chromatography
using AcOEt/hexane (1:1). Yield: 0.43 g (38%) as yellow oil.
1H NMR (CDCl3): 1.24 (t, 3H, CH2CH3, J ) 7.1 Hz); 1.24
(t, 3H, CH2CH3, J ) 7.1 Hz); 3.71 (c, 2H, CH2CH3, J ) 7.1
Hz); 3.66 (c, 2H, ′CH2CH3, J ) 7.1 Hz). 13C NMR (CDCl3): 13.1
(CH2CH3); 13.8 (CH2′CH3); 37.7 (′CH2CH3); 39.8 (CH2CH3);
152.7 (3-CdO); 165.9 (5-CdO). Anal. (C6H10N2SO2 ) C, H,
N, S.
1
0.75 g (35%) as white solid; mp 60-61 °C. H NMR (CDCl3):
3.2 (s, 3H, CH3); 4.8 (s, 2H, CH2-Bn); 7.31-7.45 (m, 5H,
arom.).13C NMR (CDCl3): 31.4 (CH3); 46.0 (CH2-Bn) 128.2;
128.6; 128.8; 135.1 (C arom.); 155.2 (3-CdO); 165.6 (5-CdO).
Anal. (C10H10N2SO2) C, H, N, S.
4-Eth yl-2-p r op yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (3). Re-
agents: Ethyl isothiocyanate (0.56 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), propyl isocyanate (0.62 mL, 6.5
mmol). Conditions: rt, 9 h. Purification: silica gel column
chromatography using AcOEt/hexane (1:3). Yield: 0.44 g (36%)
Isolation of (23): The filtrate was evaporated. Purification:
silica gel column chromatography using CH2Cl2/hexane (1:1).
Yield: 0.08 g (8%) as yellow solid; mp 91-95 °C. 1H NMR
(CDCl3): 4.52 (s, 2H, ′CH2-Bn); 5.10 (s, 2H, CH2-Bn); 7.31-
7.52 (m, 10H, arom.). 13C NMR (CDCl3): 50.1 (CH2-Bn); 54.3
(′CH2-Bn); 128.1; 128.4; 128.9; 135.4 (C arom.); 127.1; 127.4;
1
as orange oil. H NMR (CDCl3): 0.9 (t, 3H, CH2CH2′CH3, J )

First Non-ATP Competitive GSK-3â Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1297
128.4; 138.6 (′C arom.); 148.1 (3-CdS); 169.0 (5-CdO). Anal.
(C16H14N2S2O) C, H, N, S.
anate (0.38 mL, 6.5 mmol). Conditions: rt, 6 h. Isolation:
filtration of reaction mixture. Purification: recrystallization
from ethanol. Yield: 0.47 g (30%) as white solid; mp 175-178
4-Ben zyl-2-eth yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (9). Re-
agents: Benzyl isothiocyanate (0.86 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), ethyl isocyanate (0.51 mL, 6.5 mmol).
Conditions: rt, 10 h. Purification: silica gel column chroma-
tography using CH2Cl2/hexane (1:1) and CCTLC using CH2-
1
°C. H NMR (CDCl3): 3.25 (s, 3H, CH3); 7.32-7.44 (2 d, 4H,
arom., J ) 8.9 Hz). 13C NMR (CDCl3): 31.7 (CH3); 128.4; 129.6;
131.2; 135.1 (C arom.); 152.3 (3-CdO); 165.0 (5-CdO). Anal.
(C9H7ClN2SO2) C, H, N, S.
1
Cl2. Yield: 0.39 g (25%) as yellow oil; H NMR (CDCl3): 1.22
4-(4-F lu or op h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (16). Reagents: 4-Fluorophenyl isothiocyanate (1.1 g,
6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl isocy-
anate (0.38 mL, 6.5 mmol). Conditions: rt, 8 h. Isolation:
filtration of reaction mixture. Purification: recrystallization
from ethanol. Yield: 0.37 g (25%) as white solid; mp 178-180
°C. 1H NMR (CDCl3): 3.25 (s, 3H, CH3); 7.13-7.36 (m, 4H,
arom.). 13C NMR (CDCl3): 31.7 (CH3); 116.3; 129.1; 160.9;
164.2 (C arom.); 152.5 (3-CdO); 165.2 (5-CdO). Anal. (C9H7-
FN2SO2) C, H, N, S.
(t, 3H, CH2′CH3 , J ) 7.2 Hz); 3.7 (c, 2H, ′CH2CH3, J ) 7.2
Hz); 4.8 (s, 2H, CH2-Bn); 7.32-7.44 (m, 5H, arom.). 13C NMR
(CDCl3): 13.7 (CH2′CH3 ); 39.9 (′CH2CH3); 45.8 (CH2-Bn);
128.1; 128.6; 128.8; 135.1 (C arom.); 152.6 (3-CdO); 165.9
(5-CdO). Anal. (C11H12N2SO2) C, H, N, S.
2,4-Diben zyl-1,2,4-th ia d ia zolid in e-3,5-d ion e (10). Re-
agents: Benzyl isothiocyanate (0.86 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), benzyl isocyanate (0.80 mL, 6.5
mmol). Conditions: rt, 9 h. Isolation: filtration of reaction
mixture. Purification: recrystallization from ethanol. Yield:
2-Meth yl-4-(4-tr iflu or om eth ylp h en yl)-1,2,4-th ia d ia zo-
lid in e-3,5-d ion e (17). Reagents: 4-Trifluoromethylphenyl
isothiocyanate (0.66 g, 3.25 mmol), 35% HCl (1.5 mL), KMnO4
(0.25 g), methyl isocyanate (0.14 mL, 3.25 mmol). Conditions:
rt, 10 h. Isolation: solvent evaporation. Purification: silica gel
column chromatography using CH2Cl2. Yield: 0.05 g (5%) as
white solid; mp 164-166 °C. 1H NMR (CDCl3): 3.32 (s, 3H,
CH3); 7.51-7.73 (2 d, 4H, arom., J ) 8.6 Hz). 13C NMR
(CDCl3): 31.7 (CH3); 123.8 (CF3); 126.5; 127.4; 130.9; 147.6
(C arom.); 153.1 (3-CdO); 164.9 (5-CdO). Anal. (C10H7F3N2-
SO2) C, H, N, S.
1
0.57 g (30%) as white solid; mp 78-80 °C. H NMR (CDCl3):
4.7 (s, 2H, ′CH2-Bn); 4.8 (s, 2H, CH2-Bn); 7.32-7.55 (m, 10H,
arom.). 13C NMR (CDCl3): 46.0 (CH2-Bn); 48.8 (′CH2-Bn);
128.3; 128.5; 128.7; 134.4 (′C arom.); 128.3; 128.8; 128.9; 135.2
(C arom.); 153.1 (3-CdO); 165.9 (5-CdO). Anal. (C16H14N2SO2)
C, H, N, S.
4-P h en yl-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-d ion e (11)
a n d 2,4-Dip h en yl-5-oxoth ia d ia zolid in e-3-th ion e (22). Re-
agents: Phenyl isothiocyanate (0.78 mL, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), methyl isocyanate (0.38 mL, 6.5
mmol). Conditions: rt, 8 h. Isolation (11): filtration of reaction
mixture. Purification: recrystallization from methanol. Yield:
0.25 g (30%) as white solid; mp 174-179 °C. 1H NMR (CDCl3):
3.21 (s, 3H, CH3); 7.31-7.50 (m, 5H, arom.). 13C NMR (CDCl3):
31.7 (CH3); 127.2; 129.2; 129.4; 132.7 (C arom.); 152.7 (3-CdO);
165.3 (5-CdO). Anal. (C8H8N2SO2) C, H, N, S.
Isolation of (22): The filtrate was evaporated. Purification:
silica gel column chromatography using CH2Cl2. Yield: 0.14
g (15%) as yellow solid; mp 105-110 °C. 1H NMR (CDCl3):
6.70-7.01 (m, 5H, arom); 7.12-7.33 (m, 5H, ′arom.). 13C NMR
(CDCl3): 127.2; 128.6; 129.4; 132.7 (C arom.); 128.7; 129.2;
129.7; 146.7 (′C arom.); 152.4 (3-CdS); 169.3 (5-CdO). Anal.
(C14H10N2S2O) C, H, N, S.
4-(4-Meth ylp h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (18). Reagents: 4-Methylphenyl isothiocyanate (0.88
mL, 6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl
isocyanate (0.38 mL, 6.5 mmol). Conditions: rt, 6 h. Isola-
tion: filtration of reaction mixture. Purification: recrystalli-
zation from CH2Cl2/hexane. Yield: 0.29 g (21%) as white solid;
mp 182-184 °C. 1H NMR (CDCl3): 2.4 (s, 3 H, p-CH3-Ph);
3.25 (s, 3H, CH3); 7.20-7.34 (m, 4H, arom.). 13C NMR
(CDCl3): 21.1 (p-CH3-Ph); 31.7 (CH3); 126.7; 130.0; 130.3;
139.3 (C arom.); 152.9 (3-CdO); 165.3 (5-CdO). Anal. (C10H10N2-
SO2) C, H, N, S.
4-(4-Meth oxylp h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-
3,5-d ion e (19). Reagents: 4-Methoxylphenyl isothiocyanate
(0.89 mL, 6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl
isocyanate (0.38 mL, 6.5 mmol). Conditions: rt, 8 h. Isola-
tion: filtration of reaction mixture. Purification: recrystalli-
zation from CH2Cl2/hexane. Yield: 0.44 g (30%) as white solid;
4-(4-Br om op h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (12). Reagents: 4-Bromophenyl isothiocyanate (1.4 g,
6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl isocy-
anate (0.38 mL, 6.5 mmol). Conditions: rt, 9 h. Isolation:
filtration of reaction mixture. Purification: recrystallization
from hexano/CH2Cl2. Yield: 0.32 g (20%) as white solid; mp
1
mp 140-144 °C. H NMR (CDCl3): 3.31 (s, 3H, CH3); 3.80 (s,
1
3 H, p-CH3O-Ph); 7.02-7.32 (m, 4H, arom.). 13C NMR
(CDCl3): 31.7 (CH3); 55.5 (p-CH3O-Ph); 114.7; 125.3; 128.5;
159.9 (C arom.); 152.9 (3-CdO); 165.5 (5-CdO). Anal. (C10H10N2-
SO3) C, H, N, S.
182-184 °C. H NMR (CDCl3): 3.25 (s, 3H, CH3); 7.25-7.61
(2 d, 4H, arom., J ) 8.6 Hz). 13C NMR (CDCl3): 31.6 (CH3);
123.0; 128.6; 131.6; 132.5 (C arom.); 153.4 (3-CdO); 165.7 (5-
CdO). Anal. (C9H7BrN2SO2) C, H, N, S.
2-Meth yl-4-n aph th yl-1,2,4-th iadiazolidin e-3,5-dion e (20).
Reagents: Naphthyl isothiocyanate (1.2 g, 6.5 mmol), 35% HCl
(3.1 mL), KMnO4 (0.5 g), methyl isocyanate (0.38 mL, 6.5
mmol). Conditions: rt, 8 h. Isolation: filtration of reaction
mixture. Purification: recrystallization from methanol. Yield:
0.05 g (8%) as brown crystal; mp 191-193 °C. 1H NMR
(CDCl3): 3.31 (s, 3H, CH3); 7.49-8.02 (m, 7H, arom.). 13C NMR
(CDCl3): 31.8 (CH3); 125.4; 126.7; 127.1; 127.5; 128.7; 129.5;
129.6; 130.6; 131.6 (C arom.); 152.9 (3-CdO); 165.1 (5-CdO).
Anal. (C13H10N2SO2) C, H, N, S.
4-(3-Br om op h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (13). Reagents: 3-Bromophenyl isothiocyanate (1.4 g,
6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl isocy-
anate (0.38 mL, 6.5 mmol). Conditions: rt, 8 h. Isolation:
filtration of reaction mixture. Purification: recrystallization
from n-propanol. Yield: 0.40 g (21%) as white solid; mp 113-
116 °C. 1H NMR (CDCl3): 3.15 (s, 3H, CH3); 7.22-7.51 (m,
4H, arom.). 13C NMR (CDCl3): 31.6 (CH3); 122.5; 130.3; 130.5;
132.5; 133.7 (C arom.); 152.1 (3-CdO); 164.9 (5-CdO). Anal.
(C9H7BrN2SO2) C, H, N, S.
4-(2-Br om op h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (14). Reagents: 2-Bromophenyl isothiocyanate (0.87
mL, 6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl
isocyanate (0.38 mL, 6.5 mmol). Conditions: rt, 9 h. Isola-
tion: filtration of reaction mixture. Purification: recrystalli-
zation from n-butanol. Yield: 0.41 g (22%) as white solid; mp
4-Ben zyl-2-et h yl-5-oxo-1,2,4-t h ia d ia zolid in e-3-t h ion e
(24). Reagents: Benzyl isothiocyanate (0.86 mL, 6.5 mmol),
35% HCl (3.1 mL), KMnO4 (0.5 g), ethyl isothiocyanate (0.57
mL, 6.5 mmol). Conditions: rt, 12 h. Isolation: solvent
evaporation. Purification: silica gel column chromatography
using CH2Cl2/hexane (1:2) first and preparative thin-layer
chromatography using CH2Cl2/hexane (1:10) after. Yield: 0.04
g (3%) as yellow oil. 1H NMR (CDCl3): 1.2 (t, 3H, CH2CH3,
J ) 7.0 Hz); 4.25 (c, 2H, CH2CH3, J ) 7.0 Hz); 4.5 (s, 2H,
CH2-Bn); 7.11-7.31 (m, 5H, arom.). 13C NMR (CDCl3): 11.2
(CH2CH3); 46.1 (CH2-Bn); 56.2 (CH2CH3); 127.2; 127.3; 128.6;
1
136-140 °C. H NMR (CDCl3): 3.15 (s, 3H, CH3); 7.22-7.63
(m, 4H, arom.). 13C NMR (CDCl3): 31.8 (CH3); 123.0; 128.7;
130.6; 131.5; 132.2 (C arom.); 151.7 (3-CdO); 164.4 (5-CdO).
Anal. (C9H7BrN2SO2) C, H, N, S.
4-(4-Ch lor op h en yl)-2-m eth yl-1,2,4-th ia d ia zolid in e-3,5-
d ion e (15). Reagents: 4-Chlorophenyl isothiocyanate (1.1 g,
6.5 mmol), 35% HCl (3.1 mL), KMnO4 (0.5 g), methyl isocy-
138.3 (C arom.); 154.3 (3-CdS); 168.7 (5-CdO). Anal. (C11H12
-
N2S2O) C, H, N, S.

1298 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Martinez et al.
2-Eth yl-3-oxo-4-ph en yl-1,2,4-th iadiazolidin -5-im in e (27).
A solution of ethyl-(2-ethyl-3-oxo-4-phenyl-1,2,4-thiadiazolidin-
5-ylidene)urea (26)31 (0.06 g, 0.2 mmol) and NaOH (0.2 mL,
0.2 mmol) in 20 mL of MeOH was stirred at room temperature
for 18 h. Purification: CCTLC using AcOEt /hexane (1:1).
Yield: 0.022 g (50%) as white solid; mp: 200-202 °C. 1H NMR
(CDCl3): 1.25 (t, 3H, CH2CH3, J ) 7.14 Hz); 3.71 (c, 2H, J )
7.14); 7.25-7.42 (m, 5H, arom.). 13C NMR (CDCl3): 14.7 (CH2-
CH3); 39.1 (CH2CH3 ); 121.0; 126.1; 129.9 138.0 (C arom.); 164.5
(3-CdO); 170.7 (5-CdNH). Anal. (C10H11N3OS) C, H, N, S.
phosphorylation of the peptide PANKTPPKSPGEPAK by the
protein kinase C (PKC) using phosphatidyl serine as stimulat-
ing agent. The method followed is the same described above
for GSK-3â.
PKC purified from rat brains following the method described
by Walsh35 and 1 mM of substrate were used in a total volume
of 12 µL in 25 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 0.5 mM
EGTA, 0.5 mM EDTA, 7 mM CaCl2, and 100 mg/mL PS buffer
solution containing 10 µM (γ-32P)ATP.
In these conditions, none of the compounds essayed gathered
in Table 1 showed any inhibition of PKC.
Biologica l Eva lu a tion . Ma ter ia ls: The recombinant rab-
bit glycogen synthase kinase 3â (catalog number G 1663) and
the catalytic subunit of cAMP dependent protein kinase (P
2645), phenylmethylsulfonyl fluoride, benzamidine, leupeptin,
aprotinin, pepstatin, sodium fluoride, 2-mercaptoethanol, EDTA,
EGTA, and MES were obtained from Sigma (St. Louis, MO).
Cyclin dependent kinase 1-cyclin B, (catalog number 14-103)
was purchased from Upstate Biotechnology, Inc. (New York).
Histone H1 and Ro 31-8220 were supplied by Calbiochem.
DEAE-cellulose and phosphocellulose were from Whatman
Ltd. (London). ATP was purchased from Boehringer-Mann-
heim. (γ-32P)ATP was purchased from Amersham. Alkaline
phosphatase was obtained from Roche (catalog number 713023,
Germany). Stathmin was purified from bovine brain es-
sentially as described by Belmont and Mitchinson34 with some
modifications. As an additional step in the purification,
proteins were chromatographed on DEAE-cellulose, before the
HPLC step. The pellet was dissolved in 50 mL of 10 mM Tris-
HCl, 1 mM EDTA, 15 mM 2-mercaptoethanol, pH 7.5 (buffer
A), and applied to DEAE-cellulose, equilibrated in the same
buffer. Stathmin was eluted with a gradient from 0 to 1 M
NaCl.
Ca sein Kin a se II (CK-II) In h ibition : The phosphorylat-
ing activity of this enzyme against stathmin has been mea-
sured using CK-II purified from bovine brains, following the
method described by Alcazar,36 with 10-15 µg of substrate
(stathmin) in a total volume of 12 µL of 50 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 1 mM EGTA, and 1 mM EDTA buffer
solution, at 37 °C, containing 15 µM (γ-32P)ATP and 100 ng of
purified CK-2. After the reaction took place, it followed the
same method described for PKA.
In these conditions, none of the compounds essayed gathered
in Table 1 showed any inhibition of CK-II.
Cyclin Dep en d en t Kin a se 1-Cyclin B In h ibition : The
potential inhibition of this enzyme was evaluated by determin-
ing the histone H1 phosphorylation by cyclin dependent kinase
1-cyclin B.
UBI (an enzyme composed of an equal amount of the Cdk-1
and the 45 kDa cyclin B subunit, catalog number 14-103) and
10-15 µg of substrate (histone H1) were used in a 12 µL total
volume of buffer solution containing 15 µM ATP and 0.5 µCi
of (γ-32P)ATP. The kinase activity (50 ng/reaction) was per-
formed in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.01% Brij
35, and 1 mM EGTA buffer, at 37 °C. After 20 min incubation
at 37 °C, 4 µL aliquots of the supernatant were spotted onto
2 × 2 cm pieces of Whatman P81 phosphocellulose paper, and
20 s later, the filters were washed four times (for at least 10
min each time) in 1% phosphoric acid. The dried filters were
transferred into scintillation vials, and the radioactivity was
measured in a liquid scintillation counter
GSK-3â In h ibition : GSK-3â enzyme (Sigma) was incu-
bated with 15 µM of ATP, 0.2 µCi of [γ-32P]ATP, GS-1 as
substrate,32 and different concentrations of the test compound.
GSK-3 activity was assayed in 50 mM Tris-HCl, pH 7.5, 10
mM MgCl2, 1 mM EGTA, and 1 mM EDTA buffer, at 37 °C,
in the presence of 15 µM GS-1 (substrate), 15 µM [γ-32P]ATP
in a final volume of 12 µL. After 20 min incubation at 37 °C,
4 µL aliquots of the supernatant were spotted onto 2 × 2 cm
pieces of Whatman P81 phosphocellulose paper, and 20 s later,
the filters were washed four times (for at least 10 min each
time) in 1% phosphoric acid. The dried filters were transferred
into scintillation vials, and the radioactivity was measured in
a liquid scintillation counter. Blank values were subtracted,
and the GSK-3â activity was expressed in picomoles of
phosphate incorporated in GS-1 per 20 min or in percentage
of maximal activity. The IC50 (concentration at which a
50% of enzyme inhibition is shown) values are gathered in
Table 1.
In these conditions, none of the compounds essayed gathered
in Table 1 showed any inhibition of Cdk-1/cyclin B.
Ack n ow led gm en t. These investigations were fi-
nancially supported by NeuroPharma S.A. and by
CICYT (project no. SAF 99/98).
Refer en ces
(1) Brion, J . P.; Anderton, B. H.; Authelet, M.; Dayanandan, R.;
Leroy, K.; Lovestone, S.; Ocatve, J . N.; Pradier, L.; Touchet, N.;
Tremp, G. Neurofibrillary Tangles and Tau Phosphorylation.
Biochem. Soc. Symp. 2001, 67, 81-88.
(2) Alonso, A.; Zaidi, T.; Novak, M.; Grundke-Iqbal, I.; Iqbal, K.
Hyperphosphorylation Induces Self-Assembly of Tau into Tangles
of Paired Helical Filaments/Straight Filaments. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 6923-6928.
P r otein Kin a se A (P KA) In h ibition : The potential
inhibition of this enzyme is evaluated by determining the
stathmin phosphorylation by the protein kinase A (PKA). The
stathmin was purified following the procedure described by
Belmont and Mitchinson.34
(3) Anderton, B. H.; Betts, J .; Blackstock, W. P.; Brion, J . P.;
Chapman, S.; Connell, J .; Dayanandan, R.; Gallo, J . M.; Gibb,
G.; Hanger, D. P.; Hutton, M.; Kardalinou, E.; Leroy, K.;
Lovestone, S.; Mack, T.; Reynolds, C. H.; Van Slegtenhorst, M.
Sites of Phosphorylation in Tau and Factors Affecting Their
Regulation. Biochem. Soc. Symp. 2001, 67, 73-80.
(4) Larner, A. J . Tau Protein as a Therapeutic Target in Alzheimer’s
Disease and Other Neurodegenerative Disorders. Expert Opin.
Ther. Pat. 1999, 9, 1359-1370..
(5) Castro, A.; Martinez, A. Inhibition of Tau Phosphorylation: A
New Therapeutic Strategy for the Treatment of Alzheimer’s
Disease and Other Neurodegenerative Disorders. Expert Opin.
Ther. Pat. 2000, 10, 1519-1527.
(6) Imahori, K.; Uchida, T. Physiology and Pathology of Tau Protein
Kinases in Relation to Alzheimer’s Disease. J . Biochem. 1997,
121, 179-188.
Purified PKA (Sigma, catalytic subunit from bovine heart
(p 2645)) and 10-15 µg of substrate (stathmin) were used in
a 12 µL total volume of buffer solution containing 15 µM ATP
and 0.2 µCi of (γ-32P)ATP. The cAMP kinase protein (100 ng/
reaction) was performed in 50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 1 mM EGTA, and 1 mM EDTA buffer, at 37 °C. After
20 min incubation at 37 °C, 4 µL aliquots of the supernatant
were spotted onto 2 × 2 cm pieces of Whatman P81 phospho-
cellulose paper. After 20 s later, the filters were washed four
times (for at least 10 min each time) in 1% phosphoric acid.
The dried filters were transferred into scintillation vials, and
the radioactivity was measured in a liquid scintillation counter
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